Fig. 5

Uptake of fluorescent probes by PS4 cells after 20 h of exposure to P4–9. Cells were treated with P4–9 as described in Fig. 4, and after the washing steps, they were allowed to grow in fresh MH medium without the peptide for 20 h at 37 °C. Then, cells were harvested by centrifugation, washed with PBS and suspended in the same buffer. Partition of NPN (a) or propidium iodide (b, c, d, e, f, g) into this cell suspension, labeled as +P4–9 in the figure, was analyzed by fluorimetry and confocal microscopy, respectively. In parallel, duplicate suspensions not treated with P4–9 (labeled as -P4–9 in the figure) were subjected to the same procedure. To record fluorescence emitted by PI (c, f), suspensions were exposed to an excitation wavelength of 488 nm and images were digitally merged (d, g) with those of the same field obtained using visible light (b, e). Results in A are the means ± standard deviation of three independent experiments performed in triplicate. Data were analyzed using two-sample t-test and statistical differences were extremely significant (***p < 0.001). Images shown in lower panels are representative of those visualized in three independent experiments. Percentage of PI-positive cells in 5D and 5G was 2% + 1.52 and 19% + 2.06, respectively. Data (i.e. three independent fields from each experiment) were analyzed using a two-sample t-test and statistical differences were extremely significant (***p < 0.001)